Process for immunochromatography with colloidal particles

ABSTRACT

The present invention relates to improved specific binding assay methods, kits and devices utilizing chromatographically mobile specific binding reagents labelled with colloidal particles. Specific binding reagents labelled with colloidal particles such as gold and selenium may be subjected to rapid chromatographic solvent transport on chromatographic media by means of selected solvents and chromatographic transport facilitating agents. Further, impregnation of solid substrate materials with labile protein materials including colloidal particle and enzyme labelled reagents in the presence of meta-soluble proteins provides for the rapid resolubilization of such materials which have been dried onto such substrate materials.

BACKGROUND

[0001] The present invention relates generally to assay devices andspecifically to those devices making use of chromatographic techniquesin conducting specific binding assays. According to one aspect of theinvention, methods and devices are provided utilizing colloidal particlelabelled specific binding materials which are chromatographically mobileand capable of producing visually detectable signals. According toanother aspect of the invention, methods and devices are providedutilizing labelled specific binding materials including colloidalparticle labelled materials and enzyme labelled materials which aredried onto a chromatographic medium in the presence of a meta-solubleprotein and are capable of being rapidly resolubilized in the presenceof an appropriate solvent such as the sample or a chromatographictransport solvent.

[0002] Immunological assays have proven to be of great value in avariety of clinical applications. Such assays depend upon specificbinding reactions between immunoglobulins (antibodies) and materialspresenting specific antigenic determinants (antigens). Antibodies bindselectively with ligand materials presenting the antigen for which theyare specifically reactive and are capable of distinguishing the ligandfrom other materials having similar characteristics.

[0003] Because the results of immunological and other specific bindingreactions are frequently not directly observable, various techniqueshave been devised for their indirect observation. Such techniquesinvolve labelling of one of the members of the specific binding pairwith a radioisotope, chromophore, fluorophore or enzyme label.Radiolabels, chromophores and fluorophores may be detected by the use ofradiation detectors, spectrophotometers or the naked eye. Where membersof a specific binding pair are tagged with an enzyme label, theirpresence may be detected by the enzymatic activation of a reactionsystem wherein a compound such as a dyestuff, is activated to produce adetectable signal.

[0004] There are three well known types of immunological specificbinding assays. In competitive binding assays, labelled reagents andunlabelled analyte compounds compete for binding sites on a bindingmaterial. After an incubation period, unbound materials are washed offand the amount of labelled reagent bound to the site is compared toreference amounts for a determination of the analyte concentration inthe sample solution. A second type of immunological assay is known as asandwich assay and generally involves contacting an analyte samplesolution to a surface presenting a first binding materialimmunologically specific for that analyte. After a wash step, a solutioncomprising a labelled second binding material specifically reactive withthe analyte to be detected is then added to the assay. The labelledsecond binding material will bind to any analyte which is itself boundto the first binding material. The assay system is then subjected to awash step to remove any labelled second binding material which failed tobind with the analyte. The amount of labelled material remaining maythen be determined and will be indicative of the amount of analytepresent in the sample. While the term sandwich assay is frequentlyunderstood to relate to immunological assays wherein the first and thelabelled reagent materials are both antibodies or are both antigens suchthat the “sandwich” is of the form antibody/antigen/labelled antibody, abroader definition of the term sandwich-type assay is understood asincluding other types of three component assays including what aresometimes referred to as “indirect sandwiches”, which may be of the formantigen/antibody/labelled (anti-immunoglobulin) antibody.

[0005] A third type of immunological assay is the agglutination assaywhich is exemplified by well-known assays for blood antigens and serumtypes. Immunological reactivity between antibodies within serum andantigens presented on red blood cell surfaces is indicated by theformation of a three dimensional cross-linked network of antigen (redblood cells) and antibodies. The agglutination of the serum/red bloodcell mixture results in the formation of a macroscopic pellet in thetesting well which can be visible to the naked eye.

[0006] These various immunoassay procedures were originally performed as“liquid phase” assays in apparatus such as test tubes whereantigen/antibody conjugates were centrifuged and precipitated. Morerecently, methods have been developed wherein antibodies or antigens arecoated onto the surface of microtiter wells and reactions are carriedout in solution in such wells. Methods have also been developed forcarrying out “solid phase” assays wherein immunological reactions arecarried out in solution on solid substrates including those which areporous or fibrous materials. According to such procedures, porouscarrier materials are fashioned into strips or other forms to whichantibodies or antigens are immobilized by adsorption, absorption orcovalent bonding. Sample materials containing an analyte specificallyreactive with the immobilized member of the binding pair are applied tothe carrier material where the analyte is immobilized by reaction withits corresponding binding pair member. The non-reacted sample materialsare then removed by a washing step after which, in the case of asandwich-type assay, a labelled reagent is applied to the carriermaterial which is capable of reaction with and immobilization by theimmobilized analyte. The carrier material is then washed in order thatthe presence of the labelled reagent, and hence the analyte, may bedetected.

[0007] Modifications of such “solid phase” assays are known wherein oneor more of the sample components or reagents is moved by means ofchromatographic solvent transport. U.S. Pat. No. 4,168,146 to Grubb, etal., discloses porous test strips to which antibodies have beenimmobilized. The strips are then contacted with measured amounts ofaqueous solution containing the analyte antigen. Antigen moleculeswithin the test solution migrate by capillary action throughout the teststrip, but because the bound antibodies retard the migration of theantigens for which they are specific, the extent of migration of theantigen molecules over a fixed time period is a function of the antigenconcentration in the test solution. The antigen-containing areas of thediagnostic device are then indicated by the addition of enzyme orfluorescent chromophore labelled antibodies.

[0008] U.S. Pat. No. 4,517,288 to Giegel, et al. discloses methods forconducting solid phase immunoassays on inert porous materials. Thepatent discloses immunologically immobilizing a binding material withina specified zone of the porous material and applying the sample to thezone containing the immobilized binding material. An enzyme labelledindicator material which will bind with the analyte is then applied tothe zone where it will become immobilized in an amount correlated to theamount of analyte in the zone. A solvent is then applied to the centerof the zone to chromatographically remove the unbound labelled indicatorfrom the zone so that the amount of labelled indicator remaining in thezone may be measured.

[0009] Of interest to the present invention are the disclosures of theDeutsch, et al., U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537 whichrelate to immunological and other types of specific binding assayswherein reagents are transported by chromatographic solvent transport.According to one embodiment, a radiolabelled competitive binding assaykit comprises a strip capable of transporting a developing liquid bycapillarity having a first zone for receiving a sample, a second zoneimpregnated with a first reagent capable of being transported by thedeveloping liquid and a third zone impregnated with a second reagent. Inaddition, the devices comprise a measuring zone and a retarding elementwhich may be either the second reagent or the material of the strip. Thefirst reagent is capable of reacting with one of the group consisting of(1) the sample, (2) the sample and the second reagent, or (3) the secondreagent in competition with the sample, to form a product in an amountdependent on the characteristic being determined. A sample is contactedwith the first zone and the strip is then dipped into the developingliquid to bring about transport of the sample and the first reagent toform the reaction product. The retarding element slows transport ofeither the product or the first reagent (the moving reagent) tospacially separate the two and the amount of the moving element is thenmeasured at the measurement location.

[0010] The Deutsch, et al., patents relate to methods wherein reagentslocated on the chromatographic material are mixed with the samplematerial and other reagents during the course of chromatographictransport. Such mixing is not detrimental to and may even be desirablefor competitive binding assays. It may, however, be undesirable forsandwich-type binding assays where it is necessary to prevent contactbetween non-analyte sample materials and labelled specific bindingreagents.

[0011] Of interest to the present invention is the disclosure of U.S.Pat. No. 4,452,901 to Gordon which relates to the use of porousnitrocellulose supports for immobilization of proteins. It is disclosedthat such nitrocellulose sheets may be utilized in immunoassayprocedures if the residual binding capacities of the nitrocellulosesheets are saturated by blocking treatment with one or more types ofproteins, different from those immobilized and not cross-reactive withany of the antibodies subsequently used in the assay.

[0012] Of further interest to the background of the invention are thedisclosures of Gordon, EPO Application 63,810, published Nov. 3, 1982,relating to devices for conducting immunological assays. The devicesconsist of a porous solid support containing a preselected array ofdelimited adsorption areas of antigens, antibodies or both, whereinresidual adsorption sites on the substrate are saturated by proteinblocking agents such as bovine serum albumin. Porous solid supports areselected from a variety of natural and synthetic polymers andderivatives but are preferably nitrocellulose sheets 0.1 mm thick withpore size between about 0.15 μm and about 15 μm. Antigens or antibodiesare applied to the porous solid support by direct contact followed byincubation with blocking agents. Assays for detection of unknownantigens or antibodies are then carried out through use of labelledantibodies which may also be anti-immunoglobulin antibodies.

[0013] Also of particular interest to the present application is thedisclosure of co-owned and copending U.S. patent application Ser. No.912,878 filed Sep. 29, 1986 by Gordon, et al., which is herebyincorporated by reference and which relates to devices for conductingspecific binding assays utilizing the sequential chromatographictransport of analyte and reagent materials. Wash and addition steps areinherently carried out and liquid “microcircuitry” can be programmed tocarry out a variety of multistep procedures and to avoid the prematuremixing of sample materials and reagents. Preferred blocking solutionsfor treatment of the strip materials include include 1% LB gelatin(Inotech, Wohlen, Switzerland) in TBS solution comprising (0.15 M NaCl,0.02 Tris-HCl, pH 7.6) or 3% bovine serum albumin (BSA) solution inphysiological saline.

[0014] Specifically, the Gordon, et al., sequential transportapplication relates to devices which comprise a test strip for thedetection of an analyte in a sample comprising a length ofchromatographic material having the capacity for rapid chromatographicsolvent transport of non-immobilized reagents and reactive samplecomponents by means of a selected chromatographic solvent. The stripincludes a first end at which chromatographic transport begins, a secondend at which chromatographic transport ends and a plurality of zonespositioned between the two ends. The zones include a first zone(impregnated with a first reagent which is mobile in the solvent andcapable of reaction with, and immobilization against solvent transportby the analyte when the analyte is in immobilized form), a second zone(for receiving the sample suspected of containing an analyte) and athird zone (positioned downstream of the first zone and impregnated witha second reagent which is immobilized against solvent transport and iscapable of selective reaction with the analyte so as to render theanalyte in an immobilized form in the third zone). The device is furthercharacterized in that after the sample is received in the second zoneand upon the first end being dipped into the chromatographic solvent,the relative mobility of the analyte and the first reagent or the siterelationship between the second and third zones is such that the analyteis disposed and immobilized against solvent transport at the third zoneprior to the first reagent reaching the third zone, whereby interferingsample components and non-analyte components of the sample which arereactive with the first reagent are cleared from the third zone bychromatographic solvent transport prior to transport of the firstreagent to the third zone. The presence of the first reagent immobilizedat the third zone may be detected by means of enzyme, radioisotope orother labels. The device is particularly suited for use with enzymelabelled reagents as enzyme substrates and indicator dye reagents may beincorporated on separate zones on the strip and transported to the thirdzone in an appropriate sequence by chromatographic transport.

[0015] Of interest to the present invention are those referencesrelating to the use of dispersions of colloidal particles inimmunological assay procedures. Frens, Nature, 241, 20-23 (1973)discloses methods for the preparation of mono-disperse gold sols ofvarious particle sizes through the reduction of gold chloride withaqueous sodium citrate. Variation in the concentration of sodium citrateduring the nucleation of the particles may be used to vary the particlesize of the resulting sols. Sols of mono-dispersed gold particles aredisclosed having particle sizes ranging from 16 nm to about 150 nm andexhibiting colors ranging from orange to red to violet over that range.

[0016] Romano, et al., Immunochemistry, 11, 521-22 (1974) discloses thelabeling of immunoglobulins with colloidal gold particles for use inimaging human red blood cell antigens by means of electron microscopy.The gold sol, which has an average particle diameter of about 3 nm, hasa tendency to flocculate but is stabilized by the presence of eitherhorse serum or BSA.

[0017] Geoghegan, et al., J. Immuno. Meth., 34, 11-21 (1980) disclosesthe coating of colloidal gold particles with immunoglobulins for use inpassive agglutination procedures. The reference (at page 14) disclosesthe resuspension of centrifuged pools of gold labelled immunoglobulinswith 0.01 M phosphate buffered saline (PBS) (pH 7.2) containing 1%polyethylene glycol (PEG). The reference also notes that while thegold-protein complexes do not aggregate during centrifugation, they areoften subject to non-specific aggregation in the presence of anyserially diluted protein in a microtiter plate.

[0018] Surek, et al., Biochem. and Biophys. Res. Comm., 121, 284-289(1984) discloses the use of protein A labelled colloidal gold particlesfor the detection of specific antigens immobilized on nitrocellulosemembranes. According to the procedure, an electrophoresis gel is blottedonto a nitrocellulose filter which is then treated with a 2% solution ofBSA in PBS to prevent non-specific binding. The filter is treated withdiluted antiserum or preimmune serum and washed with PBS-BSA. The stripis then incubated for 30 to 60 minutes with protein A conjugated withcolloidal gold which detects the presence of bound antibodies. Excessunbound colloidal gold particles are then removed by several shortbuffer washes.

[0019] Leuvering, U.S. Pat. No. 4,313,734 discloses the use of metal solparticles as labels for in vitro determination of immunologicalcomponents in an aqueous test medium. Specifically disclosed areimmunoassay test kits for the detection of antigens or antibodiesemploying one or more labelled components obtained by coupling thecomponent to particles of an aqueous sol dispersion of a metal, metalcompound or polymer nuclei coated with a metal or metal compound havinga particle size of at least 5 nm. According to one example, an assay forhuman placental lactogen (HPL) is conducted with the use of rabbitanti-HPL antibodies which have been labelled with gold particles.Unlabelled rabbit anti-HPL antibodies are coated onto the walls ofmicrotiter plate wells by incubation with BSA solution and phosphatebuffer to which merthiolate has been added. Standard solutions of HPLare added to the wells and were incubated for 2 hours at roomtemperature. A solution consisting of rabbit anti-HPL antibodies whichhas been conjugated with gold particles having diameters between 45 and70 nm is added to the wells and incubated at room temperature overnight.The wells are then washed and light absorption measured with asmall-volume spectrophotometer.

[0020] Hsu, Anal. Biochem. 142, 221-225 (1984) discloses the use ofimmunogold marker systems for blot immunoassay procedures wherein serialdilutions of purified tobacco mosaic virus (TMV) are electrophoresed ina polyacrylamide gel and are then electrotransferred to nitrocellulosefilter sheets. The nitrocellulose sheets are baked to stabilize bindingand treated with 5% normal goat serum or in 0.05% Tween 20 in PBS toblock nonspecific antibody binding. The filter is incubated overnight at4° C. with rabbit anti-TMV antibodies diluted in blocking solutionfollowed by washing in PBS and the antigen-antibody complex is thendetected by soaking the filter in gold-labelled goat anti-rabbit IgG inblocking solution. According to the procedure as little as 8 ng of theTMV protein is detectable with about 30 minutes exposure to thegold-labelled IgG. The reference also discloses that agents such aspolyethylene glycol, polyvinylpyrrolidone and bovine serum albumin canenhance the stability of gold markers. The use of Tween 20 to preventnonspecific binding of protein on nitrocellulose is disclosed along withthe observation that 0.05% Tween 20 in PBS can be used in the stainingprocedure without disturbing the specificity of the gold-IgG complexes.Normal goat serum is identified as a preferred blocking agent in lightof its tendency to adsorb gold particles that may dissociate from theprobe during storage.

[0021] Moeremans, et al., EPO Application No. 158,746 discloses the useof colloidal metal particles as labels in sandwich blot overlay assays.Specific binding materials specifically reactive with the analyte to bedetected are applied to nitrocellulose strips and dried. Protein bindingsites on the strip are then blocked by means of treatment with bovineserum albumin, gelatin, polyethylene glycol or Tween 20. Analytecontaining sample material is then applied to the strip and incubatedfor 2 hours. The treated strip is washed and air-dried and incubated for2 hours with a specific binding agent which has been labelled withcolloidal metal particles. According to one example, anti-tubulinantibodies are detected by gold particle labelled reagents producing apink-reddish color (20 nm particles) or a purplish color (40 nmparticles). The assays are disclosed to have a sensitivity on the orderof 5 ng/μl.

[0022] Of interest to the present invention is the disclosure of Hoye J.Chromatog., 28, 379-384 (1967) relating to chromatographic purificationof radiochemicals. The application of paper chromatography, thin layerchromatography and high voltage electrophoresis techniques are disclosedto move gold ions with varying degrees of success but are generallyunsuitable for transporting colloidal gold particles.

[0023] The use of polymerized dye materials in colloidal form forspecific binding assays is also known. Of interest to the presentapplication is the disclosure of Hirschfeld, U.S. Pat. No. 4,166,105which relates to labelled specific binding reagents reactive withspecific antigens prepared by linking fluorescent dye molecules toanalyte specific antibodies through polymers comprising reactivefunctional groups. Also of interest to the present application is thedisclosure of Henry, U.S. Pat. No. 4,452,886 which relates to specificbinding reagents comprising antigens or antibodies linked to awater-soluble polymer consisting essentially of between 40 and 600chromophoric or fluorescent group containing monomers.

SUMMARY OF THE INVENTION

[0024] The present invention provides improved specific binding assaymethods, kits and devices utilizing chromatographically mobile labelledmaterials. According to one aspect of the invention, methods and devicesare provided utilizing colloidal particle labelled specific bindingmaterials which are chromatographically mobile and capable of producingvisually detectable signals. According to another aspect of theinvention, methods and devices are provided utilizing labelled specificbinding materials including colloidal particle labelled materials andenzyme labelled materials which are dried onto a chromatographic mediumin the presence of a meta-soluble protein and are capable of beingrapidly resolubilized in the presence of an appropriate solvent such asthe sample or a chromatographic transport solvent.

[0025] It has been discovered that specific binding materials labelledwith colloidal particles such as gold may be subjected to rapidchromatographic transport on a chromatographic medium by means ofselected solvents and chromatographic transport facilitating agents andthat use of chromatographic solvent transport assay techniquessignificantly reduces the time required for the binding reaction of acolloidal particle labelled material with its specific binding partneras compared with conventional methods. It has also been discovered thatimpregnation of solid substrate materials with labile proteins includinglabelled and unlabelled materials in the presence of an aqueous mediumcontaining meta-soluble proteins such as casein allows the rapidresolubilization of such proteins which have been dried onto suchsubstrate materials. Such resolubilized labelled materials may then bemaintained in a liquid solution for carrying out liquid phase assays orelectron microscopy. The drying of labile proteins in the presence ofmeta-soluble proteins thus provides an inexpensive means of storing suchproteins, including colloidal particle labelled reagents, in a stableand convenient form from which they might then be resolubilized andsubjected to chromatographic solvent transport. Alternatively, where thesolid substrate material is a chromatographic medium, the labileproteins may be solubilized and chromatographically transported to carryout specific binding assay procedures.

[0026] Accordingly, the invention provides improved specific bindingassay devices, kits and methods for determining the presence or amountof a substance in a sample. The colloidal particle labelled assaysprovide a visually detectable signal and do not require the use ofmaterials such as radioisotopes or enzyme labels with the attendantrequirement for detection equipment or addition of enzyme conjugates andindicator dyes. Means for conducting both competitive binding and directbinding (sandwich-type) assays are provided by the invention. Preferredassay methods and kits are provided for determining the presence oramount of a substance in a sample whereby a colloidal particle labelledmaterial and a chromatographic transport facilitating agent are mixedwith the sample. A chromatographic medium comprising one or morereaction sites impregnated with one or more reagents useful for carryingout the assay is then contacted with the mixture of sample, colloidalparticle labelled material and chromatographic transport facilitatingagent in order to chromatographically transport the sample and labelledmaterial along the chromatographic medium and carry out the desiredassay.

[0027] The invention also provides assay methods and devices whereinlabelled materials including colloidal particle labelled materials andenzyme labelled materials are impregnated and dried onto a reaction siteon a chromatographic substrate material in the presence of meta-solubleproteins. The chromatographic mediums of the devices may comprise one ormore additional reaction sites where chemical reactions per se need nottake place but where additional materials may be deposited orimmobilized or where analyte substance containing sample materials maybe deposited. The chromatographic medium is contacted with achromatographic solvent which solubilizes and transports along themedium the colloidal particle labelled specific binding material as wellas the sample substance and other optional materials and reagents. Theaffinity of the immobilized specific binding reagent is such that itefficiently captures the labelled material in the flowing material suchthat the labelled binding component is accumulated in the zone. Wherethe labelled material is a colloidal particle labelled material, animportant advantage is provided by the present invention in that thebinding affinity of the immobilized reagent may be such that it iscapable of capturing a labelled component in the flowing chromatographicstream in such a way that the labelled binding component is accumulatedin the capture zone and is clearly discernable over the backgroundstream of non-concentrated colloidal particle labelled material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIGS. 1a, 2 a, 3 a, 4 a and 5 a are front plan views of fivedifferent forms of the test devices of the present invention;

[0029]FIGS. 1b, 2 b, 3 b, 4 b and 5 b are cross-sectional views of thetest devices shown in FIGS. 1a, 2 a, 3 a, 4 a and 5 a, respectively,taken along lines 1 b-1 b, 2 b-2 b, 3 b-3 b, 4 b-4 b and 5 b-5 b;

[0030]FIGS. 1c, 2 c and 3 c are cross-sectional views of the testdevices shown in FIGS. 1a, 2 a and 3 a, respectively, in contact with avolume of sample material and indicator solution;

[0031]FIGS. 4c and 5 c are cross-sectional views of the test devicesshown in FIGS. 4a and 5 a, respectively, in contact with a volume ofchromatographic solvent; and

[0032]FIG. 6 is a cross-sectional view of the test device of FIG. 4ataken along lines 6-6.

DETAILED DESCRIPTION

[0033] The present invention provides improved immunological and otherspecific binding assay methods, kits and devices utilizingchromatographically mobile labelled specific binding materials.

[0034] Colloidal particle labelled specific binding materials are highlysusceptible to aggregation and are thus generally incapable of beingrapidly and efficiently transported on chromatographic media accordingto chromatographic solvent transport assay methods. The invention isbased on the discovery that specific binding materials labelled withcolloidal particles, and particularly with colloidal particles largerthan about 1 nm in diameter, which are especially subject toaggregation, may be subjected to rapid chromatographic transport onchromatographic media by means of selected solvents and chromatographictransport facilitating agents. While materials labelled with colloidalparticles and particularly those less than about 1 nm in diameter may becapable of chromatographic transport without the presence of thechromatographic transport facilitating agents of the present invention,the use of such agents assists in the rapid chromatographic transport ofall colloidal particle labelled reagents of the invention. As a relateddiscovery, it has been found that chromatographic solvent transport ofcolloidal particle labelled materials significantly reduces the timerequired for the binding reaction of those materials with their specificbinding partners as compared with conventional methods. Whileconventional immunoassay procedures such as those of Leuvering, U.S.Pat. No. 4,313,734 teach the incubation of colloidal particle labelledspecific binding materials for from 1 hour to overnight (16 hours), thechromatographic methods of the present invention generally provide forthe rapid completion of transport and specific binding reactions in lessthan about 5, and preferably less than about 2, minutes.

[0035] As another aspect of the present invention, it has furthermorebeen discovered that labile protein materials including colloidalparticle, enzyme or other labelled materials which are impregnated anddried onto solid substrate materials in the presence of selectedmeta-soluble protein materials may be rapidly solubilized by means ofsuitable solvents. Where the solid substrate material is achromatographic medium, the labelled materials may be rapidlyresolubilized and transported along the chromatographic medium by meansof selected chromatographic solvents. As a consequence of thisdiscovery, specific binding assay devices are provided wherein labelledmaterials including colloidal particle labelled specific bindingmaterials are incorporated in dry stable form on the device and only thesample material and a chromatographic solvent need be added forconducting an assay.

[0036] According to the invention, kits may be produced and specificbinding assay methods may be practiced for analysis of a substrate in asample according to a method employing a solution comprising a colloidalparticle labelled material. The method also employs a chromatographicmedium having capillarity and the capacity for chromatographic solventtransport of non-immobilized reagents and reactive sample components bymeans of a selected chromatographic solvent including a reaction siteincluding an immobilized reagent capable of binding a member selectedfrom the group consisting of the substance to be analyzed and thecolloidal particle labelled material. The method comprises (a)contacting the sample to be analyzed to the chromatographic medium, (b)chromatographically transporting on said chromatographic medium saidcolloidal particle labelled material whereby at least a portion of saidcolloidal particle labelled material is chromatographically transportedto the reaction site for binding thereto, and (c) determining thedetectable response produced by said colloidal material at the reactionsite as an indication of the presence or amount of the substance in thesample.

[0037] According to preferred embodiments of the invention, the affinityof the immobilized reagent and the concentrations of reagents and samplematerials may be selected by one of skill in the art such that thecolloidal particle labelled material is accumulated at the reaction siteand is detectable over the background stream of the non-concentratedcolloidal particle labeled material. Where this is not the case, thechromatographic medium may be subjected to a wash or rinse step toremove the unbound labelled material. Such wash steps may also beinherently carried out according to the procedures of co-owned andcopending U.S. patent application Ser. No. 912,878.

[0038] It is also to be understood that said sample to be analyzed, saidcolloidal particle labelled material, said chromatographic solvent andother solvents and reagents may be mixed with each other according tovarious possible combinations prior to their contacting thechromatographic medium and that these mixtures and various componentsmay be contacted to the chromatographic medium in various sequences aswould be apparent according to the skill in the art. It is to be furtherunderstood that the chromatographic solvent may be replaced by thesample or by the solution containing the colloidal particle labelledmaterial where these materials are capable of transporting the colloidalparticle labelled material to the reaction site at which the reagent isimmobilized. It is also to be understood that the chromatographicsolvent may inherently be used according to the methods of co-owned andcopending U.S. patent application Ser. No. 912,878 to wash unreactedlabelled materials and other non-immobilized sample components from thereaction site at which the reagent is immobilized. When the labelledmaterial is contacted to the chromatographic medium at the reaction siteat which the reagent is disposed, the chromatographic solvent transportmay be used to accelerate the binding reaction between the colloidalparticle labelled material and other specific binding reagents as wellas wash non-immobilized labelled material from the zone.

[0039] A preferred embodiment of the invention is that wherein theindicator solution additionally comprises a chromatographic transportfacilitating agent and it and the sample are mixed and contacted to thechromatographic medium to provide for chromatographic transport of theanalyte substance and the labelled specific binding material. Accordingto other embodiments, the sample material and the colloidal particlelabelled material may be contacted to one or more reaction sites on theassay device upstream of the reaction site at which the reagent isimmobilized and the chromatographic medium is contacted withchromatographic solvent to transport the sample and the labelledmaterial to the reaction site at which the second reagent isimmobilized.

[0040] Sandwich-type assays may be practiced according to the methodwherein the colloidal particle labelled material is capable ofparticipating in a specific binding reaction with the analyte substance.Competitive binding assay methods may also be practiced wherein thecolloidal particle labelled material is capable of participating in aspecific binding reaction with the immobilized reagent. Kits may beproduced and the method may also be practiced wherein thechromatographic medium comprises a second reaction site impregnated witha second reagent which is immobilized against solvent transport and iscapable of selective reaction with the colloidal particle labelledmaterial to render it in an immobilized form in the second reaction sitewhere it may be detected. In sandwich-type assays, the presence ofcolloidal particle labelled material at the second reaction site acts asa control and confirms the reactivity of the first reagent. Incompetitive binding assays, the presence of colloidal particle labelledmaterial at the second reaction site indicates the degree of competitionbetween the analyte substance and the immobilized reagent.

[0041] Other methods and devices are provided according to the inventionwherein labelled specific binding materials including, but not limitedto, colloidal particle labelled and enzyme labelled materials areincorporated on the chromatographic medium of an assay device in a dryform which may be rapidly resolubilized and chromatographicallytransported along the medium by selected chromatographic solvents. Suchspecific binding assay devices comprise a chromatographic medium havingcapillarity and the capacity for chromatographic solvent transport ofone or more non-immobilized reagents and reactive sample components bymeans of a selected chromatographic solvent. The devices also comprise afirst reaction site impregnated with a dried solution of a labelledmaterial in the presence of a meta-soluble protein wherein the labelledmaterial is capable of rapid solubilization and chromatographic solventtransport in the solvent and a second reaction site at which isimmobilized a reagent capable of binding with a member selected from thegroup consisting of the analyte substance and the labelled material. Thedevice may be used by (a) contacting the sample with the chromatographicmedium; (b) solubilizing the labelled material and chromatographicallytransporting at least a portion of the labelled material to the secondreaction site for binding thereto; and (c) determining the detectableresponse produced by said labelled material at the second reaction siteas an indication of the presence or amount of the substance in thesample.

[0042] It is to be understood that the sample to be analyzed may bemixed with the chromatographic solvent and that the chromatographicmedium may be contacted to the mixture of both. It is to be furtherunderstood that the chromatographic solvent may be replaced by saidsample, said sample being capable of solubilizing said immobilized firstreagent and chromatographically transporting itself and said labelledfirst reagent to the second zone at which the second reagent isdisposed.

[0043] A preferred embodiment of the present invention is that whereinthe chromatographic solvent is replaced by the sample which is capableof solubilizing the dried labelled material and transporting the analytesubstance and the labelled material to the second reaction sitecontaining the immobilized reagent.

[0044] Sandwich-type assays may be practiced according to the methodwherein the labelled material is capable of participating in a specificbinding reaction with the analyte substance. Competitive binding assaymethods may also be practiced wherein the labelled material is capableof participating in a specific binding reaction with the immobilizedreagent. Devices may be produced and methods may also be practicedwherein the chromatographic medium comprises a third reaction siteimpregnated with a second reagent which is immobilized against solventtransport and is capable of selective reaction with the labelledmaterial to render the labelled material in an immobilized form at thethird reaction site where it can be detected. In sandwich-type assays,the presence of labelled material at the third reaction site acts as acontrol and confirms the reactivity of the labelled material. Incompetitive binding assays, the presence of labelled material at thethird reaction site indicates the degree of competition between theanalyte substance and the labelled reagent.

[0045] The invention also provides a method for the stable storage oflabile proteins including antibodies, antigens, and enzyme labelled andcolloidal particle labelled specific binding materials wherein theprotein materials may be rapidly solubilized by application of asuitable solvent. The method comprises drying the protein material,preferably under a stream of air, on a substrate in the presence of anaqueous medium containing a meta-soluble protein. The resulting productcomprises a solid substrate upon which is impregnated and dried a labileprotein in the presence of an aqueous medium containing a meta-solubleprotein. The substrate may generally be any solid material such as glassor plastic but is preferably a porous or fibrous matrix such as paper ornitrocellulose. The solid substrate may be in various forms such asstrips, pellets or the wall of a test tube or microtiter well. Theaqueous solution containing the meta-soluble protein, preferably casein,may optionally comprise chromatographic transport facilitating agentssuch as polyethylene glycol, gelatin, bovine serum albumin anddetergents.

[0046] Mix and Run Assay Devices (Sandwich-Type)

[0047] According to one aspect of the present invention, specificbinding assays may be conducted according to mix and run techniqueswherein an indicator solution comprising a colloidal particle labelledfirst specific binding reagent dissolved in a chromatographic transportfacilitating agent is mixed with the analyte substance containingsample. Assay devices according to the invention are then dipped in themixture of sample and indicator solution which is chromatographicallytransported to a first zone where a second reagent has been immobilized.According to one embodiment of a sandwich-type assay procedure, thefirst and second reagents are capable of specific binding with theanalyte. In this embodiment, the labelled first reagent binds with theanalyte after they have been mixed. The conjugate of the labelled firstreagent and the analyte is then subjected to being immobilized byreaction with the second reagent and producing a visually detectablesignal at the first zone. In the absence of analyte, labelled firstreagent will not bind at the zone and no signal will be produced there.Alternative sandwich-type assay procedures may be followed wherein athird reagent which is specifically reactive with the labelled firstreagent is immobilized at a second zone to provide a control. Stillother, competitive-type assay methods and devices are provided where theimmobilized second reagent is specifically reactive with both theanalyte and the labelled first reagent which compete for binding withthe immobilized reagent.

[0048] Referring to the drawing, FIGS. 1a, 1 b and 1 c depict a testdevice (10) for the detection of an analyte in a sample liquidcomprising a length of chromatographic substrate material (11) with afirst end (14) at which chromatographic solvent transport begins, asecond end (15) at which chromatographic solvent transport ends and afirst zone (13) impregnated with a second reagent which is immobilizedagainst solvent transport and is capable of selective reaction with theanalyte so as to render the analyte in an immobilized form. The devicefurther comprises an inert support strip (12) to which the length ofchromatographic material (11) is affixed.

[0049] According to a procedure for use of the device (10), a quantityof the sample to be tested is mixed with an indicator solutioncomprising a colloidal particle labelled first reagent and achromatographic transport facilitating agent. The quantity andconcentration of the chromatographic transport facilitating agent in theindicator solution added to the sample is selected such that it preventsaggregation and provides for rapid chromatographic solvent transport ofthe colloidal particle labelled specific binding reagent. Insandwich-type mix and run assays, the labelled first reagent willreact-to specifically bind with the analyte. The test device (10) isthen dipped at its first end (14) into a container (16) containing themixture of sample and indicator solution (17) and the sample/indicatorsolution mixture containing the labelled first reagent/analyte conjugateprogresses through the chromatographic material (11) to the first zone(13). The second specific binding reagent immobilized at the first zone(13) is also specifically reactive with the analyte and will react withthe analyte or with first reagent/analyte conjugate to immobilize it atthe first zone (13). The chromatographic solvent transport is such,however, that labelled first reagent which is not conjugated with theanalyte along with other sample and indicator solution materials whichare not immobilized at the first zone (13) are transported away fromthat zone. Chromatographic solvent transport continues until thesample/indicator solution mixture is depleted or until thesample/indicator solution front reaches the second end (15) of thedevice.

[0050] Analyte present in a sample will bind with the labelled firstreagent and will be chromatographically transported to the first zone(13) where it will be immobilized by a specific binding reaction withthe second reagent. Where sufficient analyte is present in a sample, thenumber of colloidal particles thus immobilized at the first zone (13)will be such as to produce a visually detectable signal. Of course, ifno analyte is present in the sample, neither analyte nor labelled firstreagent will be immobilized at the first zone, and no signal will beproduced.

[0051] Mix and Run Assay Devices (Competition-Type)

[0052] The device (10) according to FIG. 1 may also be modified toperform competition-type specific binding assays. Specifically, thecolloidal particle labelled first reagent may be selected to competewith the analyte for binding with the immobilized second reagent.Referring to the drawing, FIG. 1 depicts a test device for performingmix and run competition-type assays. The device itself and the identityof the immobilized second reagent is the same in the competition-typeassay as in the sandwich-type assay. The only difference in the assaykits lies in the identity of the colloidal particle labelled firstreagent material. In the sandwich-type assay kits of the invention, thelabelled first reagent is specifically reactive with the analyte whilein competition-type assays the labelled first reagent is a specificbinding analogue of the analyte to be assayed and is specificallyreactive with the immobilized second reagent in competition with theanalyte.

[0053] According to a procedure for use of the device (10), a quantityof the sample to be tested is mixed with an indicator solutioncomprising a colloidal particle labelled first reagent in the presenceof a chromatographic transport facilitating agent. The test device (10)is then dipped at its first end (14) into a container (16) filled withthe mixture of sample and indicator solution (17). The sample/indicatorsolution mixture containing the labelled first reagent and the analyteprogresses through the chromatographic substrate material (11) to thefirst zone (13). The labelled first reagent and the analyte then competeto bind with the second specific binding reagent immobilized at thefirst zone (13). The chromatographic solvent transport is such, however,that analyte and the colloidal particle labelled first reagent materialswhich do not bind specifically with the immobilized second reagent areremoved from the first zone (13) by the chromatographic solvent.Chromatographic solvent transport will continue until the quantity ofsample/indicator solution is depleted or until the solution frontreaches the second end (15) of the device.

[0054] The quantity of analyte present in the sample will determine theamount of labelled first reagent which binds at the first zone (13).Adjustments of the quantity and/or binding affinity of the labelledfirst reagent can be made in order to determine the quantity of analytepresent in the sample.

[0055] Alternative Mix and Run (Sandwich-Assay)

[0056] According to another aspect of the present invention, assaydevices and kits are provided for performing sandwich-type assays whichcomprise a control function and which provide a positive or negativesignal for the detection of a particular analyte. Referring to thedrawing, FIG. 2 depicts a test device (20) comprising a length ofchromatographic substrate material (21) with a first end (25) at whichchromatographic solvent transport begins, and a second end (26) at whichchromatographic solvent transport ends. The device also comprises afirst zone (23) which may be separated and broken up into two or moreareas and which is impregnated with a second reagent which isimmobilized against solvent transport and is capable of selectivereaction with the analyte so as to render the analyte in an immobilizedform. The device (20) also comprises a second zone (24) which isimpregnated with a third reagent which is immobilized against solventtransport and is capable of selective reaction with the colloidalparticle labelled first reagent.

[0057] The first and second zones (23) and (24) may be shaped such as toprovide a signal of distinctive shape when one but not the other or whenboth comprise an immobilized reagent. For example, the shapes of thefirst and second zones (23) and (24) are such in FIG. 2 that when labelis immobilized at the second zone (24) only a minus (−) sign isindicated. When, on the other hand, label is immobilized at both thefirst (23) and second (24) zones a plus (+) sign is indicated.

[0058] It is preferred that the first zone impregnated with a reagentspecifically reactive with the analyte to be detected be orientedessentially perpendicular to the direction of chromatographic flow. Thisis because the analyte and hence the labelled specific binding reagenttend to become immobilized at the leading rather than the trailing edgeof the zone. Where an elongated zone is oriented with its majordimension parallel to the direction of chromatographic flow, the leadingportion of the zone will trap a majority of the analyte with the resultthat the trailing end traps little analyte and the resulting visiblesignal has a shape which may be misinterpreted. By orienting the firstzone perpendicular to the direction of chromatographic flow, a strongerand more distinct positive detection signal is produced.

[0059] According to a procedure for use of the device (20) a quantity ofthe sample to be tested is mixed with an indicator solution comprising acolloidal particle labelled first reagent in the presence of achromatographic transport facilitating agent. The test device (20) isthen dipped at its first end (25) into a container (27) containing amixture of sample and indicator solution (28) and the sample/indicatorsolution containing the labelled first reagent/analyte conjugateprogresses through the chromatographic material (21) to the first (23)and second (24) zones. The immobilized second reagent material at thefirst zone (23) is specifically reactive with the analyte and willimmobilize the analyte as well as any analyte/labelled first reagentconjugate. In addition, the immobilized third reagent material at thesecond zone (24) is specifically reactive with the labelled firstreagent and will immobilize the first reagent as well as anyanalyte/labelled first reagent conjugate.

[0060] Where analyte is present in the sample, analyte/labelled firstreagent conjugate will form in the mixture of the indicator solution andsample and the conjugate will be immobilized at both the first (23) andsecond (24) zones thus, according to one embodiment, producing a plus(+) sign and positive signal. Where no analyte or less than a thresholdamount is present in the sample, the first reagent will react with thethird reagent at the second zone (24) and will be immobilized at thatzone producing a visual signal. Because no analyte is present, nothingwill be immobilized at the first zone (23) and no signal will beproduced and only a minus (−) sign will appear indicating the absence ofanalyte. The presence of the signal at the second zone (24) but not thefirst (23) in addition to indicating absence of analyte will indicatethe mobility of the labelled first reagent and will serve as a controlrelating to the utility of the assay device.

[0061] Alternative Mix and Run (Competitor-Type Assay)

[0062] According to another aspect of the present invention, assaydevices and kits are provided for performing competition-type mix andrun assays which comprise a first zone impregnated with a second reagentspecifically reactive with the analyte to be detected and the colloidalparticle labelled first reagent and one or more second zones impregnatedwith a third reagent specifically reactive with the labelled firstreagent and not with the analyte.

[0063] Referring to the drawing, FIGS. 3a, 3 b and 3 c depict a testdevice (30) for the detection of an analyte in a sample liquid (40). Thedevice (30) comprises a length of chromatographic substrate material(31) with a first end (33) at which chromatographic solvent transportbegins and a second end (34), (which is not necessarily the secondphysical end of the strip) at which chromatographic solvent transportends. The device (30) further comprises a first zone (35) impregnatedwith a second reagent which is immobilized against solvent transport andis capable of selective reaction with a member of the group consistingof the analyte and a colloidal particle labelled first reagent.Downstream of the first zone (35) is located the second zone (36) whichmay optionally comprise more than one area and which is impregnated witha third reagent which is immobilized against solvent transport and isspecifically reactive with the labelled first reagent but not with theanalyte. The device also comprises a right-hand solvent barrier means(37) and a left-hand solvent barrier means (38) which focus thechromatographic flow of material from the first end (33) to the first(35) and second (36) zones. The solvent barrier means (37) and (38) alsoeffectively lengthen the chromatographic substrate material (31) byproviding extended chromatographic transport pathways to the second end(34).

[0064] According to a procedure for use of the device (30), a quantityof the sample to be tested is mixed with an indicator solutioncomprising a colloidal particle labelled first reagent in the presenceof a chromatographic transport facilitating agent. The first reagent isa specific binding analogue of the analyte to be assayed and isspecifically reactive with the immobilized second reagent at the firstzone (35). The test device (30) is then dipped at its first end (33)into a container (39) filled with the mixture (40) of sample andindicator solution. The sample/indicator solution mixture containing thelabelled first reagent and the analyte progresses through thechromatographic material (31) to the first zone (35). The colloidalparticle labelled first reagent and the analyte then compete to bindwith the second specific binding reagent immobilized at the first zone(35). The chromatographic solvent transport is such, however, thatanalyte and labelled first reagent materials which do not bindspecifically with the immobilized second reagent are removed from thefirst zone (35) by the chromatographic solvent and are transportedtoward the second end (34) and to the second zone(s) (36). The labelledfirst reagent, which can be a mouse anti-second reagent antibody whenthe analyte to be detected is a human anti-second reagent antibody, thenreacts with the third reagent (which can be anti-mouse IgG antibodies)immobilized at the second zone (36) which is specifically reactive withthe labelled first reagent but not the analyte. The third reagent reactswith the labelled first reagent immobilizing it at the second zone (36)producing a detectable signal. The device may optionally compriseadditional “second zones” such that where an excess of labelled firstreagent is mixed with the sample material and the labelled first reagentis partially or wholly displaced from binding at the first zone (35),the degree of displacement may be determined from the extent of bindingat the second zone(s) (36). Chromatographic solvent transport willcontinue and the sample/indicator solution will progress through thedevice until the quantity of sample/indicator solution is depleted oruntil the solution front progresses around the right-hand (37) andleft-hand (38) solvent barrier means and reaches the second end (34) ofthe device.

[0065] Pre-Impregnated Labelled Specific Binding Material Devices

[0066] An alternative aspect of the present invention relates tospecific binding assay devices wherein labelled specific bindingreagents including those with colloidal particle or enzyme labels whichare capable of chromatographic solvent transport are impregnated anddried onto the chromatographic substrate materials of the devices. Ithas surprisingly been found that drying of the labelled specific bindingreagent materials in the presence of meta-soluble proteins, such ascasein, not only provides for the rapid chromatographic solventtransport of labelled materials, but also provides for the rapidresolubilization of such labelled materials impregnated and dried ontothe chromatographic substrate materials. The labelled reagents thusresolubilized are capable of being efficiently transported by means ofconventional chromatographic solvent systems and of reacting in thespecific binding assays.

[0067] The ability to impregnate chromatographic substrate materialswith the labelled specific binding reagents, which may then beresolubilized, makes possible the practice of a variety of assayprocedures which avoid the use of labelled reagent addition steps. Bothsandwich-type and competition-type assays may be conducted using thekits and strips of the present invention.

[0068] Sandwich Assay Device

[0069] Referring to the drawing, FIGS. 4a, 4 b and 4 c depict a testdevice (50) for the detection of an analyte in a sample wherein alabelled first reagent is impregnated and dried onto the device (50).The device (50) comprises a length of chromatographic substrate material(51) with a first end (54) at which chromatographic solvent transportbegins and second ends (58) at which chromatographic solvent transportends. The length of material (51) comprises a first zone (55), a secondzone (56) and a third zone (57).

[0070] Between the first end (54) and the first zone (55) is a delayingbox defined by a right-forward solvent barrier means (60), aleft-forward solvent barrier means (61) and a transverse solvent barriermeans (62). The right-forward solvent barrier means (60) and the rightedge (63) of the material define a right-hand chromatographic solventtransport pathway, and the left-forward solvent barrier means (61) andthe left edge (64) of the material define a left-hand chromatographicsolvent transport pathway. The right-hand and left-hand chromatographicsolvent transport pathways meet downstream of the first zone (55) andthe delaying box to form a center chromatographic transport pathway inwhich are located said second (56) and third (57) zones defined by aright-rearward solvent barrier means (65) and a left-rearward solventbarrier means (66). Downstream of the third zone (57), theright-rearward solvent barrier means (65) and the right edge (63) andthe left-rearward solvent barrier means (66) and the left edge (64)define chromatographic solvent transport pathways leading to second ends(58) at which chromatographic solvent transport ends.

[0071] The first zone (55) is impregnated with a labelled first specificbinding reagent which is mobile in a chromatographic solvent (68) and iscapable of reaction with and immobilization against solvent transport bythe analyte when the analyte is in immobilized form. The second zone(56) is downstream of the first zone (55) and provides a suitable sitefor receiving the sample to be analyzed. The third zone (57) isdownstream of the second zone (56) and is impregnated with a secondreagent which is immobilized against solvent transport and is capable ofselective reaction with the analyte so as to render the analyte in animmobilized form. The device further comprises an inert support strip(52) to which the length of chromatographic substrate material (51) isaffixed. The device additionally comprises a cover plate (53) which mayoptionally be transparent and may be placed over the length of thechromatographic material (51) leaving exposed the first end (54) of thematerial. The cover plate (53) defines an opening corresponding to andleaving exposed the second zone (56). A removable tab (59) covers thesecond zone (56).

[0072] According to a procedure for use of device (50) of FIGS. 4a, 4 band 4 c, the first tab (59) is removed from the device (50), a sample ofthe material to be tested is applied to the second zone (56) and theremovable tab (59) is replaced. The device (50) is then contacted at itsfirst end (54) into a container (67) of chromatographic solvent (68).The chromatographic solvent (48) then progresses through the length ofchromatographic material passing along the right-hand and left-handchromatographic solvent transport pathways to the center chromatographictransport pathway. Some of the solvent is transported upward toward thesecond zone (56) while some of the solvent is transported downwardtoward the first zone (55) solubilizing the labelled first reagent. Aportion of the chromatographic solvent (68) from the first end (54)passes between the right-forward solvent barrier means (60) andleft-forward solvent barrier means (61) into the delaying box. Thechromatographic solvent passes around the transverse solvent barriermeans (62) which delays its flow before it is transported toward thefirst zone (55). The labelled first reagent at the first zone hasalready been solubilized by solvent from the right and left-hand solventtransport pathways and the solvent progressing through the delaying boxupon reaching the solubilized first reagent starts to transport thefirst reagent toward the second zone (56). The chromatographic solvent(68) which was transported through the right-hand and left-hand solventtransport pathways then contacts the sample applied to the second zone(56) and transports the sample to the third zone (57). There, theimmobilized second reagent material selectively reacts with analytepresent in the sample so as to immobilize it. Non-analyte components ofthe sample are transported away from the third zone (57). The labelledfirst reagent is then transported to the third zone (57) where it isimmobilized against solvent transport by the analyte when any analyte isin immobilized form. Chromatographic solvent transport of theanalyte-depleted sample and first reagent continues until thechromatographic solvent (68) reaches the second end (58) of thematerial.

[0073] A variety of sandwich-type assay devices including dried labelledreagents and preferably including dried enzyme labelled and colloidalparticle labelled reagents may be produced according to the invention.It is frequently desirable to avoid premature contact of analyte andsample materials with the reagents and contact of the reagents with eachother. Thus, the relative mobility of the sample components and thevarious reagents or the site relationship between the zones may beselected such that the reagents and sample components mix at only thetimes and locations desired. Co-owned and copending U.S. patentapplication Ser. No. 912,878 discloses various methods and devices forconducting chromatographic solvent transport assays where it is desiredto avoid contact of a labelled first reagent material (such as ananti-human immunoglobulin antibody) with sample material (such as serum)prior to the time at which the analyte antibody is immobilized againstsolvent transport at a reaction zone and other non-analyte antibodiescontained in the serum sample are cleared from the third zone bychromatographic solvent transport. The use of acceleration and delaypathways can also be particularly useful in preventing drying of samplematerials or other reagents.

[0074] Competition Assay Device

[0075] The assay devices of the present invention comprisingsolubilizable specific binding reagents are also suitable for thepractice of competitive binding type assays. According to such methods,the immobilized second reagent is selected, as in sandwich-type assays,so as to specifically bind with the analyte of interest. The labelledfirst reagent, however, is selected to be a specific binding analogue ofthe analyte which will bind competitively with the immobilized secondreagent. In carrying out competition type assays according to theinvention, it is generally not necessary that the analyte and thecolloidal particle labelled reagent be prevented from contacting eachother prior to their contacting the immobilized second reagent. Thus,the device may be designed so as to mix the analyte containing sampleand the labelled first reagent. Of course, if so desired, the device maybe designed so as to prevent contact of the sample and labelled firstreagents until after their contacting the immobilized second reagent.

[0076] Referring to the drawing, FIGS. 5a, 5 b and 5 c depict a testdevice (70) for conducting competitive binding assays for detection ofan analyte in a sample wherein a labelled first reagent is impregnatedand dried onto the device (70). The device (70) comprises a length ofchromatographic substrate material (71) with a first end (74) at whichchromatographic solvent transport begins and a second end (77) at whichchromatographic solvent transport ends. The length of material (71)comprises a first zone (75) and a second zone (76). The first zone isimpregnated with a labelled first reagent in the presence of ameta-soluble protein containing anti-aggregation buffer. The second zone(76) is downstream of the first zone (75) and is impregnated with asecond reagent which is capable of a selective binding reaction withboth the analyte and the labelled first reagent so as to render theanalyte and labelled first reagent in immobilized form. The devicefurther comprises an inert support strip (72) to which the length ofchromatographic substrate material (71) is affixed. The deviceadditionally comprises a cover plate (73) which is placed over thelength of the chromatographic substrate material (71) leaving exposedthe first end (74) of the material. The cover plate (73) defines anopening corresponding to and leaving exposed the first zone (75) whichis covered by a removable tab (78).

[0077] According to a procedure for use of device (70) of FIGS. 5a, 5 band 5 c, the tab (78) is removed from the device (70), a sample of thematerial to be tested is applied to the first zone (75) and the tab (78)is replaced. The device (70) is then contacted at its first end (74)into a container (79) of chromatographic solvent (80). Thechromatographic solvent (80) then progresses through the length of thechromatographic substrate material (71) transporting the labelled firstreagent impregnated at the first zone (75) and the sample depositedthere to the second zone (76). There the analyte and labelled firstreagent compete to bind with the immobilized second reagent for whichthey are both specifically reactive. Non-analyte components as well asunbound analyte and first reagent material are transported away from thesecond zone (76) by means of the chromatographic solvent transport whichcontinues until the chromatographic solvent is exhausted or the solventfront reaches the second end (77) of the material. At the conclusion ofthe chromatographic solvent transport, the second zone (76) may beobserved to determine the presence of labelled first reagent immobilizedat that location. The presence of labelled first reagent at thatlocation may then be related to the presence of analyte in the sample.Where the first reagent is labelled with colloidal particles, itspresence at the second zone may be observed directly. Where the firstreagent is labelled with an enzyme label, other reagents such as enzymesubstrates and indicator dyes may be added to the second zone tovisualize the presence of the first reagent.

[0078] Description of the Colloidal Particles

[0079] The present invention is directed to means for improving thechromatographic transport characteristics of colloidal particles used aslabels in specific binding assays. The colloidal particles that may beused in conjunction with the methods, kits and devices of the presentinvention are those which may be used with specific binding assaysgenerally. Particularly well known is the use of colloidal metalparticles and especially colloidal gold for carrying out immunoassays.Other colloidal particles such as polymerized dye particles which mayalso be used as labels in specific assay methods such as those ofHirschfeld, U.S. Pat. Nos. 4,166,105 and Henry, 4,452,886, thedisclosures of which are hereby incorporated by reference, may now beused in chromatographic transport specific binding assays. Particularlypreferred colloidal particle labels for use with the present inventioninclude non-metal particles such as selenium, tellurium and sulfur withselenium being particularly preferred according to co-owned andcopending U.S. application Ser. No. ______ [Attorney's Docket No. 4481],the disclosure of which is hereby incorporated by reference.

[0080] Colloidal particles which are suitable as labels according to theinvention include those which may be conjugated to specific bindingreagents without interfering with the activity of such reagents or withother reagents or analytes. The particles must be detectable andpreferably produce a visually detectable signal when present inrelatively low concentrations. Particles ranging in size from about 1 nmto about 200 nm in diameter are generally suitable although both largerand smaller particles are also suitable for use according to theinvention. The methods of the invention are particularly useful withparticles larger than about 1 nm in diameter which are particularlysusceptible to aggregation. Particles larger than about 200 nm tend toexhibit diminished mobility and may tend to drop out of suspension evenin the presence of the chromatographic transport facilitating agents ofthe present invention. Particles much larger may also have theirtransport limited by the pore size of the chromatographic transportmaterial. Particles smaller than about 1 nm tend to exhibit superiorchromatographic mobility to larger particles and in some cases may notrequire the use of the chromatographic transport facilitating agents ofthe present invention. Nevertheless, particles smaller than about 1 nmtend to provide weaker signals and are thus less suitable for use inassay procedures.

[0081] Colloidal metal particles are particularly suitable as labelsaccording to the present invention and include those particles which arecomprised of metals or metal compounds including metal oxides, metalhydroxides or metal salts. Such particles generally vary in diameterfrom about 1 nm to about 200 nm with particles ranging in diameter fromabout 40 nm to about 80 nm being particularly preferred. Particles maycomprise pure metal or metal compounds but may also comprise polymernuclei coated with metal or metal compounds. Such particles aredisclosed to have properties similar to those of particles comprisingpure metal or metal compounds. Suitable metals and metal compoundsinclude those selected from the group consisting of the metals platinum,gold, silver and copper and the metal compounds, silver iodide, silverbromide, copper hydroxide, iron oxide, iron hydroxide or hydrous oxide,aluminum hydroxide, or hydrous oxide, chromium hydroxide or hydroushydroxide, lead sulfide, mercury sulphide, barium sulphate and titaniumdioxide. Preferred metal particles include those made up of gold silveror iron oxide.

[0082] Colloidal metal particles may be produced according to methodsgenerally known in the art. Specifically, Frens, Nature, 241, 20 (1973)the disclosure of which is hereby incorporated by reference disclosesmethods for the production of gold sol particles of varying sizes. Goldparticles may be produced by methods wherein a solution of gold chlorideis heated to boiling and is then mixed with a solution of sodium citrateto reduce the gold chloride. Soon after mixing of the two solutions theboiling solution turns a faint blue indicating the onset of nucleationsoon thereafter the blue color changes to red indicating the formationof mono-disperse particles. Reduction of the gold chloride is completeafter only a few more minutes of boiling. The resulting particle sizesmay be controlled by variation of the concentration of the sodiumcitrate solution. Particles comprising other metals and metal compoundsas well as particles comprising polymer nuclei may be obtained bysimilar methodologies. The colors of the visually detectable signal fromthe metal particle label is dependent upon the identity and particlesize of the metal particle. For example, colloidal gold particlesproduce colors varying from orange to red to violet depending upon theparticle size of the sol.

[0083] Non-metal colloidal particles such as those of selenium,tellurium and sulfur may be produced according to the methods ofco-owned and copending U.S. application Ser. No. ______ [Attorney'sDocket No. 4481].

[0084] Conjugation of Binding Reagents with the Colloidal Particles

[0085] The specific binding reagents of the invention may be conjugatedwith colloidal particle labels according to methods generally known inthe art. According to one general procedure, proteinaceous specificbinding reagents and colloidal particles are rapidly mixed together andare incubated in a solution to which an agent such as bovine serumalbumin or polyethylene glycol is added. The suspension is centrifugedfirst at low speed so as to remove any large aggregates and then at highspeed to produce a pellet of the reagent/colloidal particle conjugatebefore the supernatant is aspirated and removed. The pellet isresuspended in a solution containing a chromatographic transportfacilitating agent according to the invention.

[0086] The colloidal particle labels need not be conjugated directly tothe specific binding reagents but may be coated or pretreated with otherreagents. Leuvering, U.S. Pat. No. 4,313,734, the disclosure of which ishereby incorporated by reference, discloses methods by which metal solparticles may be coated with inert polymer and copolymer coatings. Themetal sol may be brought into contact with the polymer or the sol can beplaced in an environment containing one or more monomers and apolymerization reaction initiated. After coating with the inert polymerthe immunological specific binding component may be coupled to thecoating material by adsorption or covalent binding.

[0087] Description of the Meta-Soluble Proteins

[0088] As used herein, the term meta-soluble protein refers to thoseproteins which, in their native form, are hydrophobic and poorly solublein water but which when subjected to chemical treatment, as by alkalinepurification treatment, can be made more hydrophilic and thus capable offorming uniform solutions or dispersions in water. Such chemicaltreatments serve to cleave hydrophobic fatty acid groups from theprotein molecules by cleavage of ester or other linkages. This cleavageleaves carboxy and hydroxy residues on the molecule, rendering theprotein more hydrophilic at those sites. Without intending to be limitedto a single theory of the invention, it is believed that alkalinetreatment renders the meta-soluble proteins somewhat detergent-like,that is, presenting both hydrophobic and hydrophilic aspects. Chemicaltreatment, while required for practice of the invention, need not bealkaline treatment but may also be with acids, detergents or solventssuch as alcohol or urea.

[0089] The proteins, when so treated, are capable of functioning aspotent chromatographic transport facilitating agents thus preventingaggregation and inactivation of labile proteins and reagents such asenzyme and colloidal particle labelled reagents. In addition, thetreated meta-soluble proteins when dried with a labile protein material,provides for the stable storage and prevents aggregation andinactivation when maintained in a dry state while allowing the proteinmaterials to be rapidly resolubilized and utilized in chromatographictransport assays if so desired. The labile protein materials includeantibodies, antigens or other specific binding proteins including suchproteins labelled with enzymes, colloidal particles or other labels.Preferred meta-soluble proteins include materials such as casein, zeinand a non-albumin component of egg white protein with casein inconcentrations of from 1 to 5% being particularly preferred for use withthe invention. Preferred materials include vitamin free casein (SigmaChemical Co., St. Louis, Mo., catalogue No. C-3400), Zein (Sigma,catalogue No. Z-3625) and egg white protein (Sigma, catalogue No.A-5253) which comprises both the meta-soluble protein responsible forsolubilization and transport and the inactive albumin fractions. It isknown that the egg white component responsible for solubilization andtransport is not egg white albumin as the pure albumin material does notpromote resolubilization and transport.

[0090] Description of the Chromatographic Transport Facilitating Agents

[0091] As used herein, the term chromatographic transport facilitatingagents refers to those materials which prevent aggregation andinactivation of specific binding materials and reagents in solution and,further, which promote their chromatographic transport. The agents maybe liquids or may be solids, in which case they are preferably dissolvedin a solution such as a buffer salt solution. Suitable chromatographictransport facilitating agents include materials such as polyethyleneglycol, proteinaceous materials such as gelatin and bovine serum albuminand detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate(DOC) and Triton X 100. Particularly preferred is the use ofmeta-soluble protein materials such as casein. Meta-soluble proteins mayalso be used to impregnate and dry labelled reagents onto solidsubstrate materials including chromatographic substrate materials insuch a manner that the labelled reagents may be rapidly resolubilizedand transported, if desired, by means of chromatographic solventtransport.

[0092] Where the chromatographic transport facilitating agent is to bemixed with the labelled material and utilized as a component of anindicator solution with mix and run kits, it preferably comprises caseinor another treated meta-soluble protein in combination with otherchromatographic transport facilitating agents such as PEG with buffersalt solution. A particularly preferred chromatographic transportfacilitating buffer comprises 2% casein in combination with 0.1% PEG inPBS. The concentration of the components of the buffer and of theindicator solution are selected in the practice of the mix and run kitsof the invention such that for a given sample size sufficientconcentrations of the components are provided to prevent aggregation andinactivation of the labelled reagents and promote their chromatographicsolvent transport.

[0093] Colloidal particle labelled specific binding reagents in thepresence of casein containing solutions can have Rf values approaching1.0 while colloidal labelled materials in buffers containing PEG canhave Rf values approaching 0.7. Casein concentrations in suitablechromatographic transport facilitating buffers range from between about0.1% (w/v) to greater than about 5% with concentrations of about 2%being preferred. It is noted that concentrations greater than about 5%do not appear to assist the anti-aggregation or chromatographictransport facilitating qualities of the buffer while they may, however,tend to interfere with resolubilization of labelled reagents dried ontothe test strips of the invention.

[0094] Solutions comprising PEG as the only chromatographic transportfacilitating agent are suitable for practice of some aspects of thepresent invention. PEG containing buffers have Rfs as high as 0.7.Preferred PEG concentrations in suitable chromatographic transportfacilitating buffers range from about 0.05% to about 2% with about 1%being preferred. Suitable PEG polymers may have a variety of molecularweights, with molecular weights of about 20,000 being particularlypreferred.

[0095] Gelatin is generally unsuitable for use alone as achromatographic transport facilitating agent as solutions containing itprovide for an Rf of only about 0.2. It may nevertheless be useful whencombined with other anti-aggregation materials of the invention. Gelatinis generally unsuitable, however, when used in concentrations greaterthan about 2% as it contributes to the tendency to aggregate.

[0096] Buffer solutions suitable for use with the chromatographictransport facilitating agents of the invention should have a pH betweenabout 5 and 9 and should not interfere with the reactivity of theanalyte or reagents or their chromatographic transport. Preferred buffersolutions have pHs of about 7 and include buffers such as Tris and PBS.

[0097] Description of the Chromatographic Media

[0098] Chromatographic media useful with the present invention includethose chromatographic substrate materials having capillarity and thecapacity for chromatographic solvent transport of non-immobilizedreagents and reactive sample components by means of a selectedchromatographic solvent. The chromatographic substrate materials usedwith the invention are preferably in the form of strips, but it iscontemplated that they may be in other forms including, but not limitedto, particles or gel materials in a chromatographic column. While a widevariety of chromatographic strip materials such as woven and non-wovenfibrous materials used for paper chromatography are suitable for usewith the invention, the use of microporous or microgranular thin layerchromatography substrates is particularly preferred as the use of suchsubstrates improves the speed and resolution of the assays according tothe invention. The materials should preferably be inert and generallynot react physically or chemically with any of the sample components,reagents, colloidal particle labels, buffers or reaction products.

[0099] Thin layer chromatographic substrate materials particularlysuitable for use with the present invention include granular thin layerchromatographic materials such as silica or microgranular cellulose.Preferred non-granular microporous materials include microporouscellulose esters, for example, esters of cellulose with an aliphaticcarboxylic acid, such as an alkane carboxylic acid, having from 1 to 7carbon atoms, e.g., acetic acid, propionic acid, or any of the butyricacids or valeric acids. Especially preferred are microporous materialsmade from nitrocellulose, by which term any nitric acid ester ofcellulose is intended. Suitable materials include nitrocellulose incombination with any of the said carboxylic acid cellulose esters. Thus,pure nitrocellulose esters can be used as consisting of an ester ofcellulose having approximately 3 nitric groups per 6 carbon atoms. Mostpreferred is a Type SMWP material (Millipore Corp., Bedford, Mass.)which has a pore size of 5 μm.

[0100] The various chromatographic substrate materials may be used assuch in suitable shapes such as films, strips or sheets. They may alsobe coated onto or bonded or laminated to appropriate inert supportmaterials such as paper, glass, plastic, metal or fabrics. (Onepreferred inert support material is Mylar.) Such a support material notonly has the effect of providing structural support to thechromatographic substrate material but also prevents evaporation ofreagent and solvent materials during the assay procedure. Cover platesmay also be fashioned of such inert materials. Cover plates, althoughnot required for practice of the invention, lend additional structuralsupport and further prevent evaporation of reagent and solvent materialsduring the assay procedure. Such cover plates may be transparent forviewing the progression of the assay and may comprise ports for additionof sample materials, chromatographic solvent or reagents.

[0101] The chromatographic medium upon which the assays are conductedmay be any shape or size but is preferably in the form of strips ofthickness in the range of from about 0.01 mm to about 0.5 mm, and mostpreferably of about 0.1 mm. The strips may vary widely in their otherdimensions but are preferably kept fairly small in order to shorten theassay development time and minimize material usage. When the strips areextremely small in size they may be attached to a suitable handle orholder in order to aid in handling and observation of results. Stripsapproximately 3 mm wide and up to 75 mm long have been found to beparticularly suitable in the fabrication of single pathway devicesaccording to the present invention. The pore size may vary within widelimits but is preferably between about 0.05 μm and 20 μm and preferablyabout 5 μm. Pore size is limited on the lower end by the size of thetransported analytes, reagents and colloidal particle labels. If thepore size is too small, assay materials will be transported slowly ornot at all. On the higher end, pore size is limited by binding capacity.It is generally desired that chromatographic transport be rapid with thetransport and assay being completed within less than five minutes, andpreferably less than or about two minutes. Chromatographic transportshould not be so rapid that specific binding capacity is lost asreagents do not have time to specifically bind with one another. Thecombination of pore size and substrate thickness may thus be variedaccording to the characteristics of the chromatographic solvents,specific reagents, sample materials and colloidal particle labels usedin order to obtain desired properties of speed and resolution.

[0102] It is desired that in forming the strip materials of the presentinvention that any irregularities in the materials or in the edges ofthe materials which might cause uneven flow through the material beavoided. Means of fashioning the strip materials include the use of apaper cutter with a tungsten carbide rotary blade. A preferred means,however, involves the use of laser cutting which is particularlysuitable for use in mass production techniques.

[0103] Because the chromatographic media of the device is preferablychemically inert, it may have to be activated at any zone where it isdesired to immobilize a specific binding reagent against solventtransport. Various methods will be required to render the reagentimmobilized according to the particular chemical nature of the substratematerial and the second reagent. Generally, when the media isnitrocellulose or a mixed nitrocellulose ester, no special chemicallinkage is required for the immobilization of reagents. Varioustechniques may be used for other materials and reagents which includefunctionalization with materials such as carbonyldiimidazole,glutaraldehyde or succinic acid, or treatment with materials such ascyanogen bromide. Other suitable reactions include treatment with Schiffbases and borohydride for reduction of aldehydic, carbonyl and aminogroups. DNA, RNA and certain antigens may be immobilized against solventtransport by baking onto the chromatographic material. Baking may becarried out at temperatures ranging from about 60° C. to about 120° C.for times varying from about five minutes to about 12 hours, butpreferably at about 80° C. for about two hours.

[0104] Solvent Transport Barriers

[0105] Various means are known for achieving the sequential transport ofreagents and sample materials such as are disclosed in co-owned andcopending U.S. application Ser. No. 912,878.

[0106] Solvent barriers which block chromatographic flow according tothe invention may be formed by various physical or chemical etchingtechniques. Gaps of less than 0.1 mm in width have been found to preventthe flow of liquid. A preferred means for forming such barriers involvesthe use of laser etching techniques. A CO₂ laser may be used accordingto one procedure wherein Mylar backed nitrocellulose is mounted on asupporting fixture which is mounted on a computer controlled X-Y tablecapable of very close positioning tolerances. Alternatively, a beammoving mechanism may be used. Using a combination of suitable opticallenses and careful beam focusing, a laser beam spot, with a diameter ofapproximately 0.005 inches, can be focused on the nitrocellulose. Bycareful control of the laser power, a narrow path of nitrocellulose,approximately 0.005 inches wide, can either be removed from or melted tothe Mylar backing. The use of a CO₂ laser is particularly preferredbecause of the favorable coupling effect of light from the laser withthe nitrocellulose. Nevertheless, other types of lasers are suitable,provided that the laser beam wavelength produces the desired effect onthe solvent transport material. Through use of a moving beam or an X-Ytable, precision paths baffled channels or other intricate shapes may begenerated on the nitrocellulose.

[0107] Description of the Specific Binding Reagents

[0108] Specific binding reagents useful with the present inventioninclude those materials which are members of a specific binding pairconsisting of a ligand and a receptor. The ligand and receptor arerelated in that the receptor specifically binds to the ligand, beingcapable of distinguishing the ligand from other materials having similarcharacteristics. The methods, kits and devices according to the presentinvention are particularly useful in the practice of immunological assaytechniques where the specific binding reagents are antigens andantibodies. Specific binding materials such as avidin, biotin,strepatavidin and antibiotin may also be labelled with colloidalparticles and utilized in chromatographic solvent transport assaysaccording to the invention. The methods, kits and devices may also proveuseful in the practice of DNA and RNA hybridization assays and otherspecific binding assays such as those involving receptors for hormonesor other biologically active agents.

[0109] Antibodies useful in conducting the immunoassays of the presentinvention include those specifically reactive with various analytes thedetection of which in biological fluids is desired. Such antibodies arepreferably IgG or IgM antibodies or mixtures thereof, which areessentially free of association with antibodies capable of binding withnon-analyte molecules. The antibodies may be polyclonal or monoclonaland are commercially available or may be obtained by mouse ascites,tissue culture or other techniques known to the art. A typicaldescription of hybridoma procedure for the production of monoclonalantibodies may be found in Wands, J. R., and V. R. Zurawski,Gastroenterology 80:225 (1981); Marshak-Rothstein, A., et al.; J.Immunol. 122:2491 (1979); Oi, V. Y. and L. A. Herzenberg,“Immunoglobulin Producing Hybrid”, Mishell, B. B. and S. M. Shiigi(eds.) Selected Methods in Cellular Immunology, San Francisco: W. H.Freeman Publishing, 1979; and U.S. Pat. No. 4,515,893 issued to Kung, etal. The use of mixtures of monoclonal antibodies of differing antigenicspecificities or of monoclonal antibodies and polyclonal antibodies maybe desired. It is further contemplated that fragments of antibodymolecules may be used as specific binding reagents according to theinvention including half antibody molecules and Fab, Fab′ or F(ab′)₂fragments known in the art. Regardless of the particular source or typeof antibodies, however, it is preferred that they be generally free ofimpurities. The antibodies may be purified by column chromatographic orother conventional means but are preferably purified according to knownaffinity purification techniques.

[0110] Antigens and haptens useful in carrying out the immunoassays ofthe present invention include those materials, whether natural orsynthesized, which present antigenic determinants for which the analyteantibodies are specifically reactive when presented on thechromatographic strip materials of the invention. Synthesized antigensinclude those which are constructed according to conventional chemicalsyntheses as well as those constructed according to recombinant DNAtechniques. Antigen materials may also be labelled with enzymes andcolloidal particles according to the invention and used in sandwich typeassays for the detection of antibody analytes or in competition assaysfor the detection of antigen analytes.

[0111] The methods and devices according to the present invention areexpected to be useful in the practice of a wide variety of specificbinding assays including nucleic acid hybridization assays. DNA and RNAhybridization materials useful according to the present invention wouldinclude DNA and RNA polynucleotide probes having base sequencesgenerally complementary to those of analyte gene materials. The probesof the invention will generally have between about 25 and about 10,000bases and preferably between about 30 and about 5,000 bases. The probesneed not be perfectly complementary to the base sequences of analytegene materials and will generally hybridize provided about 70% orgreater homology exists between the base sequences. Conditions relatingto DNA and RNA hybridization are disclosed generally in Crosa, et al.,J. Bact. 115(3), 904-911 (1973). Polynucleotide probe materials may beobtained according to techniques well known in the art. See, e.g.,Kornberg, DNA Replication, W. H. Freeman and Co., San Francisco, 670-679(1978); Dallas, et al., J. Bacteriol. 139, 850-858 (1979) and So, etal., Nature, 277, 453-456 (1979).

[0112] Description of Blocking Agents

[0113] Blocking agents useful in preparation of devices for the specificbinding of the present invention are those agents capable of blockingexcess binding sites on the chromatographic media which might hinderchromatographic solvent transport of sample materials or reagents of theinvention. It is generally not necessary to block the chromatographicsubstrate material in the practice of mix and run assays where thespecific binding reagents are mixed with the sample material and thechromatographic solvent. Blocking of excess binding sites on thechromatographic solvent material is particularly useful, however, wherethe sample or any reagents are impregnated on the strip in the absenceof chromatographic solvent. In the construction of devices of thepresent invention, the chromatographic media is impregnated with thereagent(s) to be immobilized at the location(s) desired. Once thereagent(s) has (have) been immobilized at the desired zones, the stripis then processed so as to block excess binding sites of thechromatographic material which might interfere with chromatographicsolvent transport of other reagents or sample materials. Particularlysuitable is the use of blocking solutions comprising proteins fromsources such as casein, gelatin or total serum. Such proteins areselected to not interfere with or cross-react with reagent materials ofthe assays. Blocking of the sites may preferably be conducted by dippingthe chromatographic substrate materials in a solution of 0.2% casein inphysiological saline and air drying the strip materials. Other methodsinclude dipping in solutions of 0.1% gelatin or 0.1% BSA followed by airdrying of the substrate materials.

[0114] Description of the Chromatographic Solvent System

[0115] Kits for performing “dip and run” assays according to theinvention utilize mixtures of the sample materials and indicatorsolutions themselves for chromatographic transport of the mobileelements of the assays. Where the assay devices are not of the dip andrun format and sample materials are applied in smaller quantities tolocations not at the first end of the assay devices, chromatographicsolvents are required for transport of the various reagents and samplecomponents on the assay devices.

[0116] Suitable chromatographic solvent systems for specific bindingassays according to the invention are those capable of solubilizing theanalyte, labelled reagents and any additional reagents and materials andtransporting them on the chromatographic material. Such solvents shouldhave sufficient ionic strength to prevent electrostatic interaction ofthe transported materials with the strip material. A preferred solventfor use in immunoassay procedures according to the invention isphysiological saline solution with a pH in the neutral range. Proteinsas well as detergents such as sodium dodecyl sulfate (SDS), Triton X-100and sodium deoxycholate (DOC) may be incorporated in the chromatographicsolvent in quantities which minimize non-specific binding with the stripmaterial but not in such excess as would prevent the desired binding andimmobilization reactions. Other chromatographic solvents such as highperformance liquid chromatography (HPLC) solvents and high performancethin layer chromatography (HPTLC) solvents which favor solubilization ofproteins and other reactants and minimize binding to strip materialssuch as nitrocellulose may also be used.

EXAMPLE 1

[0117] According to this example, casein was subjected to an alkalinetreatment purification procedure. Two hundred grams of essentiallyvitamin free casein (Sigma Chemical Co., St. Louis, Mo., catalogue no.C-3400) was mixed with 800 ml of distilled water. One liter of 2 Msodium hydroxide was then added, followed by 4 ml of 30% hydrogenperoxide and the mixture was mixed overnight at room temperature.

[0118] The material was filtered through Whatman No. 1 filter paper on aBuchner funnel and approximately 94.6 ml of 100% (glacial) acetic acidwas added to the filtrate to bring the pH to 7.5. The mixture was againfiltered, as before, and approximately 220 ml of acetic acid was addedto the filtrate to bring the pH to about 4.5. The mixture was incubatedfor 30 minutes during which time a large taffy-like lump fell out ofsolution. The supernate was centrifuged at 2800 rpm in a small RC5Ccentrifuge for 30 minutes and the pellet was added to the taffy-likelump which was then washed with deionized water.

[0119] The taffy-like material was then stirred and dissolved in oneliter of 0.15 M aqueous ammonia solution. (In the event that the caseindoes not go into solution, concentrated (15 M) ammonium hydroxidesolution should be added until the pH reaches 7.5.) The casein was thenlyophilized overnight when it was completely dried, having a yield of136 grams.

EXAMPLE 2

[0120] According to this example, a colloidal gold/antibody conjugatewas produced for practice of the methods of the present invention,Siliconized glassware (Sigma silicote) was utilized throughout theprocedure wherein 200 ml of 0.01% gold chloride (HAuCl₄.3H₂O) (FisherScientific, G-54-1) was brought to a boil and 2 ml of 1% sodium citratesolution was added and the boiling is continued for 5 minutes until thecolor of the solution changes from pale yellow to purple to red. Asolution of potassium carbonate (0.02M) was added to the suspension inorder to adjust the pH to 7.6, followed by addition of goat anti-humanIgG (1 mg/ml) (Kirkegaard-Perry, Gaithersburg, Md.) such thatapproximately 10 μg IgG was added per ml of gold suspension (0.01%gold).

[0121] After one minute of incubation at room temperature, 0.1 ml of asolution of 30% bovine serum albumin in water was added to 10 ml of thegold suspension. Aggregated material was removed by centrifugation at3000 rpm in the SS34 rotor of a Sorval RC5C centrifuge for 10 minutes.The supernate was subjected to an additional centrifugation step at 6000rpm for one hour. The colloidal gold conjugate in the pellet wasresuspended in 2% bovine serum albumin in PBS (0.05 M potassiumphosphate buffer, pH 7.4, in 0.9% NaCl), the preferred conditions forliquid storage being at 4° C. The meta-soluble preparation of casein,prepared as in Example 1, was added to give a concentration of 1%immediately before application to the membrane. The conjugate was thenstored for prolonged periods in the dry state, with no loss of activityafter 6 months storage at 37° C. in the dry state.

EXAMPLE 3

[0122] According to this example, sandwich-type immunoassay devices forthe detection of rubella antibodies were constructed and used.Microporous nitrocellulose material with a thickness of approximately0.1 mm and an average pore size of 5 μm was laminated with mylar andadhesive (Monokote, Top Flite Models, Inc., Chicago, Ill.). Stripsmeasuring 1 cm by 3.5 cm were cut by high powered laser and solventtransport lanes and a delaying box were fashioned by laser etchingaccording to the general design of the device of FIG. 4. Rubella antigen(Abbott Laboratories, North Chicago, Ill.) (0.2 μl, 2500 HA titer) wasapplied to the strips at a third zone where it was immobilized and airdried. Non-specific binding sites on the chromatographic strip materialswere then blocked by incubation for 10 minutes at room temperature witha 0.1% solution of LB gelatin in water (Inotech, Wohlen, Switzerland)and the strips were allowed to dry under a stream of air. One μl of goldparticle labelled goat anti-human IgG in an anti-aggregation bufferproduced according to Example 2 was then applied to a first zone of eachstrip (adjacent to the delay box) and dried.

[0123] Positive and negative serum samples for the rubella antibody werethen applied to the second zones between the first and third zones andthe first end of the strips were dipped into a chromatographic transportsolvent comprising TBS and 1% Triton X 100. The liquid front was allowedto progress to the second ends of the devices over a period ofapproximately 2.5 minutes transporting the sample material and the goldlabelled goat anti-human IgG to the third zone. Positive sera and theimmobilization of the labelled first reagent resulted in the presence ofa red spot at the third zone. Strips tested with negative sera did notproduce a signal at the third zone.

EXAMPLE 4

[0124] In this example a mix and run sandwich-type immunoassay devicefor the detection of human chorionic gonadotropin (HCG) was constructedand used. The device which is fashioned of the same general design asthe device of FIG. 2 produces a signal confirming the presence of alabelled first specific reagent in the sample as a negative control andproduces an additional signal indicating the presence of the HCGanalyte. Microporous nitrocellulose material with a thickness ofapproximately 0.1 mm and an average pore size of 5 μm was laminated withmylar and cement (Monokote) according to the methods of Example 3.

[0125] At a first zone, the strips were impregnated with a secondreagent comprising 0.35 μl of 2 mg/ml anti-HCG polyclonal antibody inbuffered saline containing 1% sucrose. At a second zone the strips wereimpregnated with a third reagent comprising 0.45 μl of a 100 ug/ml goatanti-mouse IgG in Tris buffered saline containing 1% sucrose. The twozones were located approximately 10 mm from the first end of the stripand were arranged such that the second zone was in the form of a minus(−) sign and the first zone was located on two sides of the second zoneso that the two zones together form a plus (+) sign.

[0126] Anti-HCG antibodies (Abbott Laboratories, North Chicago, Ill.)were incubated with 1 ml of colloidal gold suspension adjusted to pH 6.6with potassium carbonate according to the method of Example 2 such thatapproximately 10 μg IgG was added per ml of gold suspension. Indicatorsolution was then prepared comprising 10 μl of colloidal gold labelledanti-HCG antibody. The gold particle labelled antibodies were added to10 μl of Tris-buffered saline containing 10% alkaline treated caseinaccording to Example 1 and 1% PEG (M.W. 20,000). The indicator solutionwas then mixed with 100 μl of a urine sample to which varying amounts ofHCG had been added.

[0127] The test strip was then contacted at its first end in the mixtureof sample and indicator solution and the liquid front was allowed torise through the zones to the second end of the strip. When the samplesolution did not contain any of the HCG antigen the mixture of sampleand indicator solution progressed through the strip. Upon contacting thesecond zone where the goat anti-mouse IgG had been immobilized thelabelled anti-HCG antibodies were immobilized by the selectiveimmunological reaction. As no HCG was present in the sample solutionthere was no specific binding with the second reagent immobilized at thefirst zone. The colloidal gold labelled reagents thus produced avisually detectable signal in the form of a minus (−) sign indicatingoperability of the reagents but absence of HCG in the sample.

[0128] When the sample solution contained HCG the labelled anti-HCGantibodies selectively bound to the analyte to form a labelledconjugate. The mixture of the sample solution and the indicator solutionwas transported by chromatographic solvent transport through the firstand second zones to the second end. Upon contacting the first zone wherepolyclonal anti-HCG antibodies had been immobilized the gold labelledantibody/HCG conjugate was immobilized by a specific binding reaction ofthe HCG antigen with the anti-HCG antibodies. At the same time, theHCG/antibody conjugates and any unconjugated labelled anti-HCGantibodies contacting the second zone were immobilized by contacting thegoat anti-mouse IgG antibodies immobilized at that zone. The colloidalgold labelled reagents thus immobilized at both the first and secondzones produced a visually detectable signal in the form of a plus (+)sign indicating the presence of HCG in the sample. The sensitivity forHCG of this format was determined to be as low as 25 milli-IU/ml.

EXAMPLE 5

[0129] In this example, a mix and run sandwich-type immunoassay for thedetection of A-polysaccharide (APS) was prepared and used according tothe methods of Example 4. According to this example, nitrocellulosestrips were prepared according to Example 4 and were treated withpolyclonal anti-APS antibodies which were immobilized at the first zone.

[0130] Rabbit polyclonal anti-APS antibodies (Abbott Laboratories, NorthChicago, Ill.) were incubated with 1 ml of colloidal gold suspensionadjusted to pH 7.2 with potassium carbonate according to the method ofExample 2 such that approximately 10 μg of anti-APS antibody was addedper ml of gold suspension. An indicator solution was then preparedcomprising 10 μl of colloidal gold labelled anti-APS antibodies. Thegold particle labelled antibodies were added to 10 μl of Tris-bufferedsaline containing 10% alkaline treated casein according to Example 1 and1% PEG. The indicator solution was then mixed with 100 μl of a swabextraction buffer for strep to which varying amounts of APS (AbbottLaboratories, North Chicago, Ill.) had been added.

[0131] The test strip was then dipped at its first end in the mixture ofa sample and indicator solution and the liquid front was allowed to risethrough the first zone to the second end of the strip. APS present inthe samples reacted with and was bound to the anti-APS antibodies in theindicator solution to form a conjugate. These conjugates were thenimmobilized at the first zone by reaction between the APS and theanti-APS polyclonal antibodies immobilized at the zone. The presence ofAPS in the swab extracted sample was indicated by the development of apurple color as a consequence of the concentration of the colloidal goldparticles at the zone. The sensitivity for APS of devices according tothis format was determined to be as low as 0.5 ng/ml APS.

EXAMPLE 6

[0132] In this example, sandwich-type immunoassay devices for thedetection of swine anti-trichina antibodies were produced according tothe general procedures of Example 3. Nitrocellulose assay strips wereprepared and were treated with partially purified trichina antigen(United States Department of Agriculture) immobilized at a detectionzone.

[0133] Colloidal selenium particles of various sizes were producedaccording to the methods of co-owned and copending U.S. patentapplication Ser. No. ______ [Attorney's Docket No. 4481] filed herewith.Various volumes (40, 80 and 150 μl aliquots) of concentrated seleniumsol were pipetted into individual vials containing 4 ml of water eachand the pH of each solution was adjusted to 7.2 by addition of 0.01 Mpotassium carbonate. To each of the vials was then added 150 μl of goatanti-swine antibody (1 mg/ml concentration) (Kirkegaard-Perry). Thesolutions were mixed and allowed to incubate for 10 minutes. A 0.5 mlaliquot of a 0.5% solution of alkaline treated casein was added to eachsolution and mixed well. Three ml aliquots of each selenium conjugatesolution were centrifuged in 1 ml portions on a TDx table centrifuge andthe pellets were combined for each conjugate after the supernatant wasdecanted off. The combined pellets of each conjugate were resuspendedwith a solution of 4% casein in 20 μl of TBS. 0.5 μl aliquots of theselenium particle labelled antibody indicator solutions were thenapplied to a first zone of each strip (adjacent to the delay box) anddried.

[0134] Positive and negative serum samples containing Trichinaantibodies were then applied to the second zones of the devices betweenthe first and third zones and the first end of the strips were dippedinto a chromatographic transport solvent comprising TBS and 1% Triton X100. The liquid front was allowed to progress to the second ends of thedevices over a period of approximately 2.5 minutes, transporting thesample material and the selenium labelled anti-swine antibodies to thethird zone. All conjugates gave visible positive signals with theconjugate utilizing 80 nm selenium particles providing the best results.All the conjugate solutions were tested against a negative control whichindicated no specific binding.

EXAMPLE 7

[0135] In this example, a mix and run sandwich-type immunoassay devicewas constructed and used according to the general procedures of Example4. Instead of incubating the anti-HCG antibodies with gold particles theantibodies were incubated with colloidal selenium particles producedaccording to the methods of co-owned and copending U.S. Ser. No. ______[Attorney's Docket No. 4481] Selenium particles of varying sizes weretested against varying concentrations of HCG. The conjugate utilizing 80nm particles gave the best results with a detection limit of 20 mIU/ml.Antibodies labelled with larger or smaller particles gave less sensitiveresults as shown in Table 1 below. All conjugate solutions were testedagainst a negative control which indicated no specific binding. TABLE 1Particle Size Detection Limit (nm) (mIU/ml)  11 500   80 20 140 50 19350 300 4000 

EXAMPLE 8

[0136] According to this example, sandwich-type immunoassay devices forthe detection of rubella antibodies were constructed and used accordingto the general procedures of Example 3. Instead of the gold particlelabelled goat anti-human IgG, however, alkaline phosphatase labelledgoat anti-human IgG (Kirkegaard-Perry) was used to detect the presenceof rubella antibodies immobilized at the second zones. One μl of 1 mg/mlof the alkaline phosphatase labelled IgG was diluted in a PBS solutioncontaining 1% alkaline treated casein and was applied to the first zoneof each strip and dried. The assay devices could then be constructed,stored for prolonged periods and used in the manner of the gold labelledassay devices of Example 3 with the exception that enzyme substrate andindicator dye reagents must be added to the test strips in order tovisualize the assay results.

[0137] Numerous modifications and variations in practice of theinvention are expected to occur to those skilled in the art uponconsideration of the foregoing descriptions of preferred embodimentsthereof. The use of colloidal particle labelled reagents inchromatographic assay techniques is of wide applicability and is notlimited to the specific examples disclosed. It is thus, well within theskill in the art to practice the present invention according to a widevariety of methods and formats. Consequently, only such limitationsshould be placed on the invention as appear in the following claims.

What is claimed is:
 1. A method for determining the presence or amountof a substance in a sample, which method comprises; (a) contacting saidsample with a chromatographic medium, said medium having at least onereaction site including an immobilized reagent capable of binding amember selected from the group consisting of the substance and acolloidal particle labelled material capable of producing a detectableresponse, (b) chromatographically transporting on said chromatographicmedium said colloidal particle labelled material whereby at least aportion of said colloidal particle labelled material ischromatographically transported to the reaction site for bindingthereto, and (c) determining the detectable response produced by saidcolloidal material at the reaction site as an indication of the presenceor amount of the substance in the sample.
 2. The method according toclaim 1 wherein said colloidal particle labelled material is transportedin the presence of a chromatographic transport facilitating agent. 3.The method according to claim 2 wherein said chromatographic transportfacilitating agent is selected from the group consisting of polyethyleneglycol, gelatin, bovine serum albumin, detergents and meta-solubleproteins.
 4. The method according to claim 3 wherein saidchromatographic transport facilitating agent is casein.
 5. The methodaccording to claim 1 wherein said colloidal particle labelled materialis capable of participating in a specific binding reaction with a memberselected from the group consisting of said substance and saidimmobilized reagent.
 6. The method according to claim 1 wherein theaffinity of the immobilized reagent is selected such that the colloidalparticle labelled material is accumulated at the reaction site and isdetectable over the background stream of non-concentrated colloidalparticle labelled material.
 7. The method according to claim 1 whereinsaid chromatographic medium comprises a second reaction site including asecond immobilized reagent which may be the same or different from saidfirst immobilized reagent and which is capable of binding with saidcolloidal particle labelled material and comprising the step, (d)determining the detectable response produced by said colloidal particlelabelled material at the second reaction site as an indication of thepresence or amount of the substance in the sample.
 8. The methodaccording to claim 1 wherein said particles have diameters ranging fromabout 1 nm to about 200 nm.
 9. The method according to claim 8 whereinsaid particles have diameters ranging from about 40 nm to about 80 nm.10. The method according to claim 1 wherein said particles are colloidalmetal particles.
 11. The method according to claim 10 wherein saidparticles are gold particles.
 12. The method according to claim 1wherein said particles are non-metal particles selected from the groupconsisting of selenium, tellurium and sulfur.
 13. The method accordingto claim 12 wherein said particles are selenium particles.
 14. A methodfor determining the presence or amount of a substance in a sample, whichmethod comprises; (a) contacting the sample with a chromatographicmedium, said medium having at least two reaction sites, the firstreaction site including a dried solution of a labelled material in thepresence of a meta-soluble protein and the second reaction siteincluding an immobilized reagent capable of binding a member selectedfrom the group consisting of the substance and said labelled material,(b) solubilizing said labelled material and chromatographicallytransporting at least a portion of said labelled material to the secondreaction site for binding thereto, and (c) determining the detectableresponse produced by said labelled material at the second reaction siteas an indication of the presence or amount of the substance in thesample.
 15. The method according to claim 14 wherein said meta-solubleprotein is selected from the group consisting of casein, zein andmeta-soluble non-albumin egg white protein.
 16. The method according toclaim 15 wherein said meta-soluble protein is casein.
 17. The methodaccording to claim 14 wherein said labelled material is capable ofparticipating in a specific binding reaction with a member selected fromthe group consisting of said substance and said immobilized reagent. 18.The method according to claim 14 wherein said chromatographic mediumcomprises a third reaction site including a second immobilized reagentwhich may be the same or different from said first immobilized reagentand which is capable of binding with said labelled material andcomprising the step, (d) determining the detectable response produced bysaid labelled material at the third reaction site as an indication ofthe presence or amount of the substance in the sample.
 19. The methodaccording to claim 14 wherein said labelled material is a colloidalparticle labelled material.
 20. The method according to claim 19 whereinsaid colloidal particles have diameters varying from about 1 nm to about200 nm.
 21. The method according to claim 20 wherein said particles havediameters ranging from about 40 nm to about 80 nm.
 22. The methodaccording to claim 19 wherein said particles are colloidal metalparticles.
 23. The method according to claim 22 wherein said particlesare gold particles.
 24. The method according to claim 19 wherein saidparticles are non-metal particles selected from the group consisting ofselenium, tellurium and sulfur.
 25. The method according to claim 24wherein said particles are selenium particles.
 26. The method accordingto claim 18 wherein said labelled material is an enzyme labelledmaterial.
 27. A method for storing labile proteins which preventsaggregation and inactivation of the proteins when maintained in the drystate wherein said proteins may be rapidly resolubilized, which methodcomprises drying the protein on a substrate in the presence of anaqueous medium containing a meta-soluble protein.
 28. The methodaccording to claim 27 wherein said meta-soluble protein is selected fromthe group consisting of casein, zein and meta-soluble non-albumin eggwhite protein.
 29. The method according to claim 27 wherein saidsubstrate comprises a porous or fibrous matrix.
 30. A product producedaccording to the method of claim 27 comprising a solid substrate uponwhich is impregnated and dried a labile protein in the presence of anaqueous medium containing a meta-soluble protein.
 31. A test device fordetermining the presence or amount of a substance in a sample, by meansof one or more specific binding reactions comprising; a chromatographicmedium having capillarity and the capacity for rapid chromatographicsolvent transport of one or more non-immobilized reagents and reactivesample components by means of a selected chromatographic solventincluding, a first reaction site including a dried solution of alabelled material in the presence of a meta-soluble protein wherein saidlabelled material is capable of rapid solubilization and chromatographicsolvent transport in said solvent, and a second reaction site includingan immobilized reagent capable of binding a member from the groupconsisting of said substance and said labelled material.
 32. The testdevice according to claim 31 wherein said meta-soluble protein isselected from the group consisting of casein, zein and meta-solublenon-albumin egg white protein.
 33. The test device according to claim 32wherein said meta-soluble protein is casein.
 34. The test deviceaccording to claim 31 wherein said labelled material is a colloidalparticle labelled material.
 35. The test device according to claim 34wherein said particles have diameters ranging from about 1 nm to about200 nm.
 36. The test device according to claim 34 wherein said particleshave diameters ranging from about 40 nm to about 80 nm.
 37. The testdevice according to claim 34 wherein said particles are colloidal metalparticles.
 38. The test device according to claim 37 wherein saidparticles are gold.
 39. The test device according to claim 34 whereinsaid particles are non-metal particles selected from the groupconsisting of selenium, tellurium and sulfur.
 40. The test deviceaccording to claim 37 wherein said particles are selenium.
 41. The testdevice according to claim 31 wherein said materials are enzyme labelledmaterials.
 42. A kit for use in specific binding assays for determiningthe presence or amount of a substance in a sample comprising; (1) asolution comprising a colloidal particle labelled material in thepresence of a chromatographic transport facilitating agent, and (2) achromatographic medium having capillarity and the capacity forchromatographic solvent transport of non-immobilized reagents andreactive sample components by means of a selected chromatographicsolvent including at least one reaction site including an immobilizedreagent capable of binding a member selected from the group consistingof said substance and said colloidal particle labelled material.
 43. Thekit according to claim 42 wherein the chromatographic transportfacilitating agent is a meta-soluble protein.
 44. A composition ofmatter comprising a colloidal particle labelled reagent and a sufficientamount of a meta-soluble protein to allow resolubilization from a driedstate and chromatographic solvent transport of said colloidal particlelabelled reagent.
 45. The composition of claim 44 wherein themeta-soluble protein is casein.